Folic acid is a precursor of folylpolyglutamates, which are coenzymes in, and potential regulators of, a large number of reactions known as one carbon metabolism. These reactions, which include metabolic cycles for the synthesis of thymidylate, purines and amino acids, are compartmentalized in the mitochondria and cytosol of tissues. This proposal is for the continuation of a series of studies aimed at investigating the role that mitochondrial folate metabolism plays in one carbon metabolism in cells and tissues. A number of approaches will be used. 1. Model cell systems defective in cytosolic and/or mitochondrial folate metabolism, cells over expressing serine hydroxymethyltransferase (SHMT) in the cytosol and mitochondria will be developed. 2. The regulation of one carbon entry into the folate pool via mitochondrial and cytosolic SHMT and the synthesis and fate of cytosolic and mitochondrial serine and glycine will be studied. The role of formate as a one carbon shuttle linking mitochondrial and cytosolic one carbon metabolism will be investigated. 3. The human cytosolic and mitochondrial SHMT genes will be characterized and nutritional factors regulating the expression of these genes identified. 4. The regulation of folate-dependent methionine synthesis and the role of the methionine cycle in sparing the nutritional need for methionine and in the retention of absorbed folate by tissues will be investigated. A methionine synthase cDNA will be cloned and the regulation of the synthase investigated. The long term goals of the project are to understand the basic mechanisms by which folate homeostasis and one carbon metabolism are regulated. The metabolic cycles under study play a central role in the de novo synthesis of the methyl group of methionine and in precursor availability for DNA and protein synthesis. Disturbances in these cycles result in megaloblastic anemia, homocysteinemia and may also be involved in the etiology of neural tube defects.